MicroRNAs (miRNAs) are involved in many diverse biological processes and a single miRNA can modulate the expression of hundreds of gene targets. Changes in miRNA expression could have profound biological impacts, leading to a variety of human diseases. To date, most miRNA expression profiling studies have relied on microarrays or high-throughput RNA sequencing (RNA-seq). For these high-throughput experiments, it is important to validate the profiling results with independent methods. Real-time PCR is considered as the gold standard for gene expression quantification and thus is ideal for validation of microarray or RNA-seq results. Furthermore, despite their wide use for miRNA profiling, there are some major limitations associated with microarrays and RNA-seq, such as the requirement of high-quality RNA samples. To address these issues, we have developed a new real-time RT-PCR method. With this new method, we have profiled the expression of cancer-related miRNAs in hundreds of archived tumor tissues, and identified novel miRNA signatures as predictive biomarkers for multiple types of cancer. Building on the success of our new method for cancer-related miRNA profiling, we propose to significantly expand the scope of the method by designing and testing new assays to encompass all known miRNAs in the human genome (Specific Aim 1). These miRNA assays will serve two main purposes: 1) As a reliable independent method for validation of high-throughput results from microarrays or RNA-seq; and 2) expression profiling of low- quality degraded RNA extracted from clinical tissues. In both cases, availability of these miRNA assays will enable researchers to correlate abnormal miRNA expression changes to human diseases by focusing on a selected set of miRNAs. To facilitate the selection of miRNA assays by end users, we also propose to establish an online database to present experimentally validated assays for all known human miRNAs (Specific Aim 2).